Antifungal agent

ABSTRACT

There is disclosed a novel compound having the formula   &lt;IMAGE&gt; (I) which exhibits antifungal activity.

This application claims priority to U.S. Provisional Patent ApplicationSer. Nos. 60/008/081, filed Oct. 30, 1995 and 60/008,956, filed Dec. 20,1995.

BACKGROUND OF THE INVENTION

The present invention relates to novel antifungal compounds,compositions and methods of use. The compound and compositions exhibitbroad spectrum antifungal activity against both human and plant fungalpathogens. Clinical treatment of human fungal infections has reliedmainly on two types of antifungal agents. These agents are amphotericinB, which is fungicidal and capable of curing fungal infections at thecost of severe side effects to the patient, and ketoconazole and otherazole agents, which exhibit fewer side effects but are only fungistatic.

Thus, there is a need for new human antifungal agents.

SUMMARY OF THE INVENTION

The present invention is directed to the compound of the formula (I):##STR2##

The compound has antimicrobial and fungicidal properties and may beuseful for controlling systemic and superficial fungal infections inhumans with fewer side effects than standard antifungal agents such asamphotericin B or ketoconazole.

The compounds are obtained by cultivation of a strain ofChaetosphaeronema, MF 6188 in the culture collection of Merck & Co.,Inc., Rahway, N.J.

DETAILED DESCRIPTION OF THE INVENTION

The compound is colorless and characterized by the following spectralproperties:

Ultraviolet Spectral Data

    λ.sub.max (MeOH): 234 nm

Infrared Spectral Data

Recorded as a thin film on ZnSe: 3245, 2929, 1717, 1624, 1439, 1206,1142, 724 cm⁻¹

Mass Spectral Data

Mass spectra were recorded on JEOL SX-102A (Electron Impact, EI, 90 eV),JEOL HX110 (Fast Atom Bombardment, FAB), and TSQ70B (LC/MS-ESI, LiquidChromatography-Electrospray Ionization) mass spectrometers. The FABspectrum was run in a matrix of dithiothreitol-dithioerythritol (20/80).The exact mass measurements were made at high resolution with Ultramark™1960 (Fomblin) as the reference compound.

HR FAB-MS Found: 546.2691 Calculated for C₂₉ H₃₉ NO₉ +H: 546.2703

NMR Spectral Data

NMR spectra were recorded in acetone-d₆ at 500 MHz (¹ H) or 125 MHz (¹³C). Chemical shifts are reported downfield from TMS (tetramethylsilane)and spectra were referenced to the solvent peak (2.05 ppm for ¹ Hspectra and 29.9 ppm for ¹³ C spectra).

¹ H NMR Spectra

δ ¹ H: 9.81(br s; 1H), 6.86(dq; 15.5, 6.6; 1H), 6.74(dq; 15.5, 1.5; 1H),6.11(ddd; 17.4, 10.0, 8.3; 1H), 5.26(ddd; 17.4, 1.5, 0.7; 1H), 5.10(dd;10.0, 1.5; 1H), 4.79(br s; 1H), 4.66 (br s; 1H), 4.05(d; 4.8; 1H),3.93(d; 10.7; 1H), 3.83(dt; 8.3, 7.2; 1H), 3.65(s; 3H), 2.80(ddd; 1H),2.61(ddd; 12.6, 4.4, 2.2; 1H), 1.80(dd; 6.6, 1.5; 3H), 1.77(m; 1H),1.73(m; 2H), 1.67(m; 2H), 1.42(m; 1H), 1.35(m; 1H), 1.28(m; 4H), 1.25(m;1H), 0.87(t; 7.2; 3H), 0.84(d; 6.7; 3H) ppm

¹³ C NMR Spectra

δ ¹³ C: 201.2(s), 196.0(s), 173.7(s), 170.6(s), 170.4(s), 152.6(s),145.0(d), 138.2(d), 135.9(s); 126.1(d), 118.6(t), 87.0(s), 77.6(d),68.7(d), 63.0(d), 51.7(q), 49.6(d), 48.5(d), 42.9(d), 41.3(d), 37.9(d),33.9(t), 32.1(t), 31.6(t), 27.8(t), 23.1(t), 21.7(q), 18.5(q), 14.3(q)ppm

The compound of this invention has antimicrobial properties and isespecially useful as an antifungal agent against both filamentous fungiand yeasts. It is useful against organisms causing systemic humanpathogenic mycotic infections such as Candida albicans, Candidatropicalis, Candida guillermondii, Candida glabrata, Cryptococcusneofromans, Aspergillus fumigatus, Candida pseudotropicalis,Saccharomyces cerevisiae, Aspergillus flavus et al. It is also usefulagainst organisms causing superficial fungal infections such asTrichoderma sp. and Candida sp. These properties may be effectivelyutilized by administering compositions containing an antifungal amountof the compound to an area, object or subject, on or in which fungi areto be controlled. Thus, compositions containing an antifungallyeffective amount of the compound and their use for the control of fungiare aspects of the present invention. An especially preferred aspect ofthe present invention are compositions in a pharmaceutically acceptablecarrier and their use for the control of mycotic infections byadministering a therapeutically effective amount of one or both of thecompounds.

The compound of the present invention is a natural product produced froma strain of Chaetosphaeronema (Coelomycetes, Deuteromycotina), MF 6188in the culture collection of Merck & Co., Inc., Rahway, N.J., which hasbeen deposited under the Budapest Treaty in the culture collection ofthe American Type Culture Collection on Nov. 29, 1995 at 12301 ParklawnDrive, Rockville, Md. 20852 and assigned accession number ATCC 74354.

The producing organism is a strain of Chaetosphaeronema (Coelomycetes,Deuteromycotina) that was isolated from forest leaf litter, collected inCuarros de Garabito, Puntarenas Province, Costa Rica.

In agar culture, colonies of the fungus exhibit the followingmorphology:

Colonies on oatmeal agar (Difco) at 25° C., 12 hr photoperiod attaining35-37 mm in 14 days, with advancing zone submerged, even, with aerialmycelium scant to velvety or woolly, dull, obscurely zonate, with someradial sectors, at first white to pale gray to dark olive gray, DawnGray, Storm Gray, Castor Gray (capitalized color names from Ridgway, R.1912. Color Standards and Nomenclature. Published by the author.Washington, D.C.), with reverse dull grayish brown, exudates absent.

Colonies on potato-dextrose agar (Difco) at 25° C., 12 hr photoperiodattaining 40-42 mm in 14 days, submerged to appressed at the margin,raised towards the center, velvety to woolly, obscurely zonate, dull,white to pale gray, finally dark olivaceous gray, Olive-Gray, StormGray, Castor Gray, Dusky Green-Gray, reverse pale ochraceous gray todark brownish gray Clay Color, Tawny-Olive, Saccardo's Umber, exudatesabsent.

Colonies on YM agar (Difco) at 25° C., 12 hr photoperiod attaining 39-40mm in 14 days, with margin submerged, even, raised, velvety, with someradial sectors, azonate, pale brownish gray to gray, finally darkbrownish gray, similar to color on oatmeal agar, reverse dark brownishgray, exudates absent. Some slight growth at 37° C., about 1-2 mm in 14days.

Mycelium composed of highly branched, simple septate, thin- to slightlythick-walled, dematiaceous hyphae characteristic of many ascomycetousfungi. In older cultures intercalary dictyochlamydospores form on theaerial hyphae, especially towards the outer edges of the Petri plates.Dictyochlamydospores elongated along the main axis of the hyphae,consisting of irregular masses of slightly thick-walled, dark cells,with individual cells up to 12 μm in diameter.

Conidiomata formed abundantly on autoclaved banana leave supported bycornmeal agar (Difco) or scattered around the edges of mature cultures(>3 weeks old) on YM agar. Conidiomata are pycnida, dark olivaceousbrown, discrete, scattered, partially imbedded in leaf tissue,subglobose, 200-400 μm in diameter, centrally papillate with a singleostiole, hairy, with hairs slightly thick-walled, septate, darkolivaceous brown, up to 200 μm long. Conidiomatal wall composed of dark,tightly woven hyphae, easily rupturing, with inner wall lined withconidiogenous cells.

Conidiogenous cells enteroblastic, phialidic, hyaline, cylindrical ortapered toward apex, with or without a slightly flared collerette,branched once or twice at the base, with periclinal thickenings atconidiogenous locus, 8-12×2.5-3.5 μm.

Conidia cylindrical, to allantoid, occasionally eccentrically curved,broadly rounded or sometimes inflated at the apices or bases, 1-septate,smooth, hyaline, without appendages, 9.5-12×3-4 μm, exuding from matureconidiomata in a whitish to translucent mass.

Pycnidial fungi, such as the producing organism, without an apparentsexual reproductive phase, are referred to the form class Coelomycetes.Following the diagnostic system of Sutton (Sutton, B. C. 1980. TheCoelomycetes. Commonwealth Mycological Institute, Kew, United Kingdom),the discrete, thin-wailed pycnidia with phialidic conidiogenous cellswould place this organism among Sutton's Blastopycnidiae. Within thisgroup of genera, the producing organism would best fit the concept ofChaetosphaeronema (Sutton pg. 408) based on the combination ofhairy-setose, ostiolate pycnidia, elongated and sparsely branchedphialides, and elongated, 1-septate conidia.

Although the invention is discussed principally with respect to thespecific strain, it is well known in the art that the properties ofmicroorganisms can be varied naturally and artificially. Thus, allstrains of the sterile fungus MF 6188, ATCC 74354 including varietiesand mutants, whether obtained by natural selection, produced by theaction of mutating agents such as ionizing radiation or ultravioletirradiation, or by the action of chemical mutagens such asnitrosoguanidine, are contemplated to be within the scope of thisinvention.

The production of the compound may be carried out by cultivating thesterile fungus MF 6188, ATCC 74354 in a suitable nutrient medium underconditions described herein until a substantial amount of antifungalactivity is detected in the fermentation broth, harvesting by extractingthe active components from the mycelial growth with a suitable solvent,concentrating the solution containing the desired component, thensubjecting the concentrated material to chromatographic separation toisolate the compound from other metabolites also present in thecultivation medium.

Broadly, the sources of carbon include glucose, fructose, mannose,maltose, galactose, mannitol and glycerol, other sugars and sugaralcohols, starches and other carbohydrates, or carbohydrate derivativessuch as dextran, cerelose, as well as complex nutrients such as oatflour, corn meal, millet, corn and the like. The exact quantity of thecarbon source which is utilized in the medium will depend, in part, uponthe other ingredients in the medium, but it is usually found that anamount of carbohydrate between 0.5 and 15 percent by weight of themedium is satisfactory. These carbon sources can be used individually orseveral such carbon sources may be combined in the same medium. Certaincarbon sources are preferred as hereinafter set forth.

The sources of nitrogen include amino acids such as glycine, arginine,threonine, methionine and the like, ammonium salt, as well as complexsources such as yeast hydrolysates, yeast autolysates, yeast cells,tomato paste, soybean meal, casein hydrolysates, yeast extract, cornsteep liquors, distillers solubles, cottonseed meal, meat extract, andthe like. The various sources of nitrogen can be used alone or incombination in amounts ranging from 0.05 to 5 percent by weight of themedium.

Among the nutrient inorganic salts, which can be incorporated in theculture media are the customary salts capable of yielding sodium,potassium, magnesium, calcium, phosphate, sulfate, chloride, carbonate,and like ions. Also included are trace metals such as cobalt, manganese,iron, molybdenum, zinc, cadmium, and the like.

Representative suitable solid and liquid production media may be seen inthe tables which follow. Also included is a representative seed medium.These, however, are merely illustrative of the wide variety of mediawhich may be employed and are not intended to be limiting.

                  TABLE 1                                                         ______________________________________                                        KF SEED MEDIUM  Trace Element Mix                                                       per liter            per liter                                      ______________________________________                                        Corn Steep Liquor                                                                         5     g     FeSO.sub.4 7.H.sub.2 O                                                                     1    g                                   Tomato Paste                                                                              40    g     MnSO.sub.4.4H.sub.2 O                                                                      1    g                                   Oat flour   10    g     CUCl.sub.2.2H.sub.2 O                                                                      25   mg                                  Glucose     10    g     CaCl.sub.2   100  mg                                  Trace Element Mix                                                                         10    ml    H.sub.3 BO.sub.3                                                                           56   mg                                                          (NH.sub.4).sub.6 Mo.sub.7 O.sub.24.4H.sub.2                                                19   mg                                                          ZnSO.sub.4.7H.sub.2 O                                                                      200  mg                                  ______________________________________                                         pH = 6.8                                                                 

                  TABLE 2                                                         ______________________________________                                        PRODUCTION MEDIUM CYS80                                                              Component                                                                              per liter                                                     ______________________________________                                               Sucrose  80          g                                                        Corn Meal                                                                              50          g                                                        (yellow)                                                                      Yeast Extract                                                                          1           g                                                 ______________________________________                                         No pH adjustment                                                         

                  TABLE 3                                                         ______________________________________                                        PRODUCTION MEDIUM STP                                                                Component                                                                              per liter                                                     ______________________________________                                               Sucrose  75          g                                                        Tomato Paste                                                                           10          g                                                        Malt Extract                                                                           5           g                                                        (NH.sub.4).sub.2 SO.sub.4                                                              1           g                                                        Soy Flour                                                                              1           g                                                        KH.sub.2 PO.sub.4                                                                      9           g                                                 ______________________________________                                         pH adjusted to 7.0 with NaOH before autoclaving                          

Of the foregoing media, the STP medium, was found to give the best yieldof the compound. In the production of the compound, generally, theculture is first grown in a seed medium and the culture growth then usedto inoculate a production medium. The production medium may be a solidmedium or a liquid medium.

The culture was maintained in sterile soil and stored at 4° C. untilready for use. The seed culture was inoculated by asepticallytransferring a small amount of the preserved soil into a 250 mlErlenmeyer flask containing 50 mls of seed medium of the followingcomposition (in g/liter); corn steep liquor, 5.0; tomato paste, 40.0;oat flour, 10.0; glucose, 10.0; and trace elements solution, 10mls/liter (consisting of, in g/liter: FeSO₄.7H₂ O, 1.0; MnSO₄.4H₂ O,1.0; CuCl₂.2H₂ O, 0.025; CaCl₂.2H₂ O, 0.1; H₃ BO₃, 0.056; (NH₄)₆MoO₂₄.4H₂ O, 0.019; ZnSO₄.7H₂ O, 0.2; dissolved in 0.6N HCl). Seedmedium was prepared with distilled water, the pH was adjusted to 6.8 byadding NaOH and the medium dispensed into 250 ml Erlenmeyer flasks andcapped with cotton plugs before being autoclaved at 121° C. for 20minutes. The seed culture was incubated at 25° C. on a gyrotory shaker(220 rpm, 5.1 cm throw) for 66 hours prior to the inoculation offermentation flasks.

The STP production medium was prepared using distilled water; 50 mlsmedium was dispensed into 250 ml Erlenmeyer flasks that were capped withcotton plugs before being autoclaved at 121° C. for 20 minutes.Production flasks were inoculated with 2.0 mls vegetative seed growthand were incubated at 25° C., on a gyrotory shaker (220 rpm, 5.1 cmthrow) for 14 to 17 days. After the incubation period, each productionflask was homogenized, extracted with 50.0 mls of methanol, shaken for30 minutes, pooled and delivered for the isolation of active compounds.

The usefulness of the compound as an antifungal agent, especially as anantimycotic agent, may be demonstrated with the compound in a brothmicrodilution assay for the determination of minimum inhibitoryconcentration (MIC) and minimum fungicidal concentration (MFC) againstfungi. In such assay against a panel of fungi selected for theirresistance/susceptibility to known compounds, animal virulence, sourceand clinical importance, the compound is found to be effective atconcentrations comparable to an established antifungal agent,amphotericin B.

In the microbroth dilution assay, microorganisms were selected bystreaking a yeast culture on Sabouraud dextrose agar (SDA) incubatingfor 24-48 hours at 35°-37° C., thereafter selected 3 to 5 characteristiccolonies and transferring to a fresh plate and incubating under similarconditions. From the regrowth, 3 to 5 colonies were selected andsuspended in 10 milliliters of YM broth (Difco) and incubated for 4hours at 35°-37° C. shaking at 225 rpm. The 4 hour broth cultures wereadjusted optically to 86% transmission resulting in a concentration of1-5×10⁶ cfu/ml which was further diluted 1:100 in YNBD (yeast nitrogenbase with 1% dextrose) to obtain a concentration of 1-5×10⁴ cfu/ml foruse as inocula.

The test compound was dissolved at 256 μg/ml in 10% DMSO and diluted 2×into the first well to achieve a concentration of 256 μg/ml at 5% DMSOin the first well. Compounds are subsequently serially diluted 2× andcell suspension is added to each well resulting in an additional 2×dilution of compound. 75 μl of said solution is delivered to each wellin column 1 of a 96-well, U-bottomed microtiter plate. The compounds incolumn 1 were then serially diluted two-fold to yield concentrationsfrom 64 μg/ml to 0.03 μg/ml.

Amphotericin B, the control compound, was prepared as a stock solutionof 512 μg/ml in 10% DMSO and 75 μl of said solution delivered to column1 of a 96-well, U-bottomed microtiter plate. The compounds in column 1were then serially diluted two-fold to yield concentrations from 128μg/ml to 0.06 μg/ml.

The plates containing the diluted compounds were then inoculated with 75μl/well of the appropriate microorganism and incubated for 48 hours at35°-37° C. with MIC (minimum inhibitory concentration) determinationscarried out after 24 hours of incubation (except Cryptococcus strainswhich are read at 48 hours). Growth and sterility controls for eachorganism and sterility checks for the compounds also were carried out.

After recording MICs at 24 hours, the microtiter plates were shakengently to resuspend the cells. A 1.5 μl sample was transferred from eachwell of the 96-well microtiter plate to a single reservoir inoculumplate containing SDA. The inoculated SDA and corresponding microtiterplates were incubated for 24 hours at 35°-37° C. For Cryptococcusneoformans, SDA plates were inoculated at 48 hours after recording MICsand incubated 48 hours before reading the MFC. MFC is the lowestconcentration of compound at which either no growth or growth of ≦4colonies occur.

    ______________________________________                                        Minimum Fungicidal Concentration (MFC)                                        Minimum Inhibitory Concentration (MIC)                                        μg/ml                                                                      Strain                MIC     MFC                                             ______________________________________                                        Candida albicans (MY1055)                                                                           <0.03   0.06                                            Candida glabrata (MY1381)                                                                           16      32                                              Candida parapsilosis (MY1010)                                                                       <0.03   <0.03                                           Candida pseudotropicalis (MY2099)                                                                   2       4                                               Candida tropicalis (MY1124)                                                                         4       8                                               Candida albicans (CLY539)                                                                           0.06    0.125                                           Candida albicans (CA2)                                                                              0.125   0.25                                            Candida tropicalis (MY1012)                                                                         1       2                                               Candida guillermondii (MY1019)                                                                      2       16                                              Candida krusei        0.06    0.5                                             Cryptococcus neoformans                                                                             <0.03   <0.93                                           (MY2061)                                                                      Cryptococcus neoformans                                                                             <0.03   <0.03                                           (MY2062)                                                                      Saccharomyces cerevisiae (MY2140)                                                                   4       0.5                                             Aspergillus fumigatus (MY4839)                                                                      <0.03                                                   Aspergillus fumigatus (MY5668)                                                                      1                                                       ______________________________________                                    

The compound is also useful for inhibiting the growth of filamentousfungi. Such use may be illustrated in the following tests withAspergillus flavus, Fusarium oxysporum, Ustilago zeae and the like.

Inocula for filamentous fungi are prepared by scraping the surface ofstock plates maintained on potato dextrose agar with a moistened steriledacron swab. The spores and mycelia are then suspended in 10 millilitersof sterile potato dextrose broth and adjusted to 70 percent transmissionat 660 nm.

The samples to be tested for production of antifungal agent are applieddirectly to the agar plates as methanol solutions. When the sample to betested is crude broth, it may be centrifuged prior to application. Theassay plates are then incubated at either 28° C. or 37° C. for 24 hours.Following incubation, the inhibition zones are measured. Growths arealso noted as to appearance. The compound is seen to effectively inhibitgrowth of the fungal organisms.

In view of the broad spectrum of activity, the product of the presentinvention either singly or as a mixture is adaptable to being utilizedin various applications of antifungal compositions. In such case,compounds may be admixed with a biologically inert carrier, generallywith the aid of a surface active dispersing agent, the nature of whichwould vary depending on whether the use is for the control of pathogensinfecting man or animals, or for control of fungi in agriculture such asin soil or plant parts, or for the control of fungi in inanimateobjects.

In compositions for medical applications, the compound may be admixedwith a pharmaceutically acceptable carrier, the nature of which willdepend on whether the composition is to be topical, parenteral or oral.

If said application is to be topical, the drug may be formulated inconventional creams and ointments such as white petrolatum, anhydrouslanolin, cetyl alcohol, cold cream, glyceryl monostearate, rose waterand the like.

For parenteral applications, the compounds may be formulated inconventional parenteral solutions such as 0.85 percent sodium chlorideor 5 percent dextrose in water, or other pharmaceutically acceptablecompositions.

Compositions for oral administration may be prepared by mixing thecomponent drugs with any of the usual pharmaceutical media, includingfor liquid preparations, liquid carriers such as water, glycols, oils,alcohols, and the like; and for solid preparations such as capsules andtablets, solid carriers such as starches, sugars, kaolin, ethylcellulose, surface active dispersing agents, generally with lubricantssuch as calcium stearate, together with binders, disintegrating agentsand the like.

These compositions are then administered in amounts sufficient to obtainthe desired antifungal effect. For medical applications, the methodcomprises administering to a subject in need of treatment atherapeutically effective antifungal amount of the compounds. Theappropriate dose will vary depending on age, severity, body weight andother conditions. For topical application, the compositions are applieddirectly to the area where control is desired. For internaladministration, the composition may be applied by injection or may beadministered orally.

For non-medical application, the product of the present invention,either alone or as a mixture, may be employed in compositions in aninert carrier which included finely divided dry or liquid diluents,extenders, fillers, conditioners and excipients, including variousclays, diatomaceous earth, talc, and the like or water and variousorganic liquids such as lower alkanols, such as ethanol and isopropanol,or kerosene, benzene, toluene and other petroleum distillate fractionsor mixtures thereof.

The following example illustrates the invention but is not to beconstrued as limiting the invention disclosed herein.

EXAMPLE I ##STR3## Isolation

The isolation procedure involved: 1) partitioning of the MEK extractbetween EtOAc and H₂ O, 2) separation of the EtOAc extracted material byopen reversed phase C-8 chromatography (60% ACN/40% H₂ O), 3) HPLCpurification on Zorbax RX-C8 (75% MeOH/25% H₂ O) and 4) a final HPLCpurification on Zorbax RX-C8 (60% ACN/40% 0.1% aq. TFA!). This procedureresulted in a >95% pure (based on nmr and HPLC) preparation of thecompound. The overall recovery of material was 19% using this procedure(400× purification).

Compound I had the spectral properties previously described.

The following examples illustrate representative compositions containingCompound I.

EXAMPLE A

1000 compressed tablets each containing 500 milligrams of Compound I areprepared from the following formulation:

    ______________________________________                                                           Grams                                                      ______________________________________                                        Compound I           500                                                      Starch               750                                                      Dibasic calcium phosphate hydrous                                                                  5000                                                     Calcium stearate     2.5                                                      ______________________________________                                    

The finely powdered ingredients are mixed well and granulated with 10percent starch paste. The granulation is dried and compressed intotablets.

EXAMPLE B

1000 hard gelatin capsules, each containing 500 milligrams of Compound Iare prepared from the following formulation:

    ______________________________________                                               Compound I      500                                                           Starch          250                                                           Lactose         750                                                           Talc            250                                                           Calcium stearate                                                                              10                                                     ______________________________________                                    

A uniform mixture of the ingredients is prepared by blending and used tofill two-piece hard gelatin capsules.

EXAMPLE C

250 milliliters of an injectible solution are prepared by conventionalprocedures from the following formulation:

    ______________________________________                                        Dextrose           12.5   grams                                               Water              250    milliliters                                         Compound I         406    milligrams                                          ______________________________________                                    

The ingredients are blended and thereafter sterilized for use.

EXAMPLE D

An ointment suitable for topical application may be prepared byintimately dispersing 13 mg of Compound I in 1 g of commerciallyavailable polyethylene/hydrocarbon gel.

EXAMPLE E

An aerosol composition may be prepared having the following formulation(per canister):

    ______________________________________                                        Compound I              24     mg                                             Lecithin NF, liquid concentrate                                                                       1.2    mg                                             Trichlorofluoromethane  4.025  g                                              Dichlorodefluoromethane 12.15  g                                              ______________________________________                                    

What is claimed is:
 1. A substantially pure compound having thestructure: ##STR4##
 2. An antifungal composition comprising anantifungal amount of the compound of claim 1 in admixture with abiologically inert carrier or diluent.
 3. A composition according toclaim 2 wherein the carrier is a pharmaceutically acceptable carrier. 4.A method for controlling fungal growth which comprises administering tothe site where growth is to be controlled, an effective amount of thecompound of claim
 1. 5. A method for combatting fungal infections inmammals which comprises administering to a region of the animalafflicted with said fungi a therapeutically effective amount of thecompound of claim
 1. 6. A process for producing the compound of claim 1which comprises aerobically cultivating a culture of ATCC 74354 in anutrient medium containing assimilable sources of carbon and nitrogenand isolating said compound therefrom.
 7. A biologically pure culture offungus (ATCC 74354) capable of producing the compound of claim 1 inrecoverable amounts.